Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: N-(2-Hydroxyethyl)-2-pyrrolidon
- Batch No.: O 2909
- Physical state: Liquid/colourless to yellow, clear
- Analytical purity: 99.7 area-%
- Stability under test conditions:The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and
the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services, Germany GmbH
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation: Male animals: 332.5 g - 358.3 g; Female animals: 188.4 g - 220.2 g
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
• Pregnant animals and their litters were housed together until PND 4. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Enrichment: wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria)
- Bedding: Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data)
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day before necropsy).
- Water: Drinking water was supplied from water bottles (ad libitum).
- Acclimation period: about 6 days

ENVIRONMENTAL CONDITIONS
The animals were housed in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20-24°C and a range
of relative humidity of 30-70%. The air change rate was 15 times per hour. There were no or only minimal deviations from these limits. The day/night
cycle was 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h. The animal room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection) before use. Walls and floor were cleaned once a week with water
containing an appropriate disinfectant.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with drinking water and subsequently intensely mixed with a magnetic stirrer until it was completely dissolved.
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the test substance preparations:
Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out prior to the start of the study. Given that test substance was completely miscible with drinking water, solutions were considered to be homogenous without further analysis. Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations.
Analytical Method:
The concentrations of the preparations were determined by LC/MS/MS in a GLP compliant study. the following set-up was used:
- HPLC System: Agilent 1100 Series HPLC system (vacuum solvent degasser, binary HPLC pump, column oven), and CTC Analytics HTC-Pal Autosampler.
- HPLC Column: Thermo Aquasil C18 column, 150 mm length, 3.0 mm i.d., 3 μm particle size.
- Injection Volume: 10 μL
- HPLC Method: Solvent A: 0.1 % formic acid in water, Solvent B: 0.1 % formic acid in methanol
Mobile Phase Composition:
Time (min); Flow rate (μL/min); % A; % B
0.00; 400; 90; 10
0.50; 400; 90; 10
0.60; 400; 5; 95
6.00; 400; 5; 95
6.10; 400; 90; 10
9.00; 400; 90; 10
- MS System Applied Biosystems API 3000 triple quadrupole LC-MS/MS system with Turbo IonSpray ESI source.
- Electrospray Ion Source Conditions: Positive ionisation:
Source temperature: 400°C
Curtain gas (CUR): 12
Nebulizer gas (NEB): 14
Ion spray voltage (IS): 5000 V
Collision gas (CAD): 4.00
Entrance potential (EP): 10 V
Resolution Q1 and Q3: Unit
- MS/MS Conditions: MRMs for quantitation of N-(2-Hydroxyethyl)-2-pyrrolidon and for confirmation (not reported): Declustering potential (DP): 26 V:
Q1 Mass; Q3 Mass; CE; CXP; Dwell
Quantitation; 130; 69; 27; 12; 500
Confirmation; 130; 112; 19; 20; 500
The [M-H]+ ion of N-(2-Hydroxyethyl)-2-pyrrolidon at 130 m/z was used as parent ion for MS/MS detection. The MS/MS transition to the daughter ion at 69 m/z was used for quantification of the analyte. For confirmation a 2nd MS/MS transition (130 m/z -> 112 m/z) was monitored.
The nominal concentration of N-(2-Hydroxyethyl)-2-pyrrolidon (PSN 04/0443-2) in water of 10.0, 3.0 and 1.0 g/100 mL were confirmed by deviations in a range of plus 9 % to 0 %.
Duration of treatment / exposure:
Male: 28 days
Female: 49 days
Frequency of treatment:
Daily, females in labor were not treated.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw /day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: By request of the sponsor
- Rationale for selecting species: The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Examinations

Parental animals: Observations and examinations:
MORTALITY:
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.

CAGE SIDE OBSERVATIONS:
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g.inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day

DETAILED CLINICAL OBSERVATIONS (DCO):
Detailed clinical observations were performed in all animals once prior to the first administration and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 x 37.5 cm wide, with side borders which are 25 cm high). The following parameters listed were assessed: 1. Abnormal behavior in “handling”, 2. Fur, 3. Skin, 4. Posture, 5. Salivation, 6. Respiration, 7. Activity/arousal level, 8. Tremors, 9. Convulsions, 10. Abnormal movements, 11. Gait abnormalities, 12. Lacrimation, 13. Palpebral closure, 14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency), 16. Assessment of the urine discharged during the examination, 17. Pupil size

BODY WEIGHT:
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION :
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7, 7 - 14 and 14 - 20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1- 4.
Food consumption was not determined in females without positive evidence of sperm during the gestation period and in females without litter duringthe lactation period.

HAEMATOLOGY:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular
hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count and Reticulocytes (RET).
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Parameter and method: Prothrombin time (Hepato Quick’s test) (HQT),

CLINICAL CHEMISTRY:
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters. Parameters:
- Enzymes: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT).
- Blood chemistry parameters: Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids
(TBA).

URINALYSIS:
The dry chemical reactions on test strips (Combur-10-test M, Roche, Mannheim, Germany) used to semi quantitatively determine urine constituents were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany). Parameters: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume.

NEUROBEHAVIOURAL EXAMINATION:
A functional observational battery was performed in 5 parental male and 5 parental female animals (with litter) per group at the end of the administration period starting at about 10.00h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: 1. Posture, 2. Tremors, 3. Convulsions, 4. Abnormal movements, 5. Impairment of gait, 6. Other findings
- Open field observations: The animals were transferred to a standard arena (50 x 50 cm wide, with side borders which are 25 cm high) and observed for at least 2 minutes. The following parameters were examined: 1. Behavior when removed from cage, 2. Fur, 3. Skin, 4. Salivation, 5. Nose discharge, 6. Lacrimation, 7. Eyes/pupil size, 8. Posture, 9. Palpebral closure, 10. Respiration, 11. Tremors, 12. Convulsions, 13. Abnormal movements/stereotypy, 14. Impairment of gait, 15. Activity/arousal level, 16. Feces excreted within 2 minutes (number of scybala discharged/appearance/consistency), 17. Urine excreted within 2 minutes (amount/color), 18. Number of rearings within 2 minutes
- Sensory motor tests/Reflexes: The animals were removed from the open field and subjected to following sensory motor or reflex tests: 1. Approach response, 2. Touch response, 3. Vision (“visual placing response”), 4. Pupillary reflex, 5. Pinna reflex, 6. Audition (“startle response”), 7. Coordination of movements (“righting response”), 8. Behavior during “handling”, 9. Vocalization, 10. Pain perception (“tail pinch”), 11. Other findings, 12. Grip strength of forelimbs and hindlimbs, 13. Landing foot-splay test
- Motor activity measurement (MA): The MA was measured on the same day as FOB was performed in 5 parental males and females (with litter) per group. The examinations were performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose, the animals were placed in clean polycarbonate cages for the time of measurement. The number of beam interrupts was counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the polycarbonate cages was selected at random. The measurement was started at about 14.00 h. On account of the measuring variant "staggered", the starting time was varied by the time needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and ended exactly 1 hour later. The animals received no food or water during the measurements. After the transfer of the last animal in each case, the room where the measurements were carried out was darkened.
Sperm parameters (parental animals):
Special attention was given on stages of spermatogenesis in the male gonads.
Litter observations:
PUP NUMBER AND STATUS AT DELIVERY:
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

PUP VIABILITY/MORTALITY:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in section “Necropsy observations”. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.

SEX RATIO:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth.

PUP CLINICAL OBSERVATIONS:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.

PUP BODYWEIGHT DATA:
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Postmortem examinations (parental animals):
GROSS PATHOLOGY:
- Clinical pathology: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using
isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were
carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and
urine was collected overnight. Urine samples were evaluated in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The following examinations were carried out in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.
- Necropsy: All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
- Weight parameters: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Epididymides and Testes
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen and Thymus.

HISTOPATHOLOGY:
The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution: 1. All gross lesions, 2. Adrenal glands, 3. Aorta, 4. Bone marrow (femur), 5. Brain, 6. Cecum, 7. Cervix, 8. Coagulating glands, 9. Colon, 10. Duodenum, 11. Eyes with optic nerve, 12. Esophagus, 13. Extraorbital lacrimal glands, 14. Epididymides (modified Davidson’s solution), 15. Femur with knee joint, 16. Heart, 17. Ileum, 18. Jejunum (with Peyer’s patches), 19. Kidneys, 20. Larynx, 21. Liver, 22. Lungs, 23. Lymph nodes (axillary and mesenteric), 24. Mammary gland (male and female), 25. Nose (nasal cavity), 26. Ovaries (modified Davidson’s solution), 27. Oviducts, 28. Pancreas, 29. Parathyroid glands, 30. Pharynx, 31. Pituitary gland, 32. Prostate gland, 33. Rectum, 34. Salivary glands (mandibular and sublingual), 35. Sciatic nerve, 36. Seminal vesicles, 37. Skeletal muscle, 38. Spinal cord (cervical, thoracic and lumbar cord), 39. Spleen, 40. Sternum with marrow, 41. Stomach (fore stomach and glandular stomach), 42. Target organs, 43. Testes (modified Davidson’s solution), 44. Thymus, 45. Thyroid glands, 46. Trachea, 47. Urinary bladder, 48. Uterus, 49. Vagina.
The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri were rinsed carefully with 0.9% NaCl solution. When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing. (SALEWSKI, E.: Färbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte; Naunyn-Schmiedeberg’s Arch. Exp. Pathol. Pharmakol. 247, 367(1964)). Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. Special attention was given on stages of spermatogenesis in the male gonads. A correlation between gross lesions and histopathological findings was attempted.

Postmortem examinations (offspring):
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.
Statistics:
- Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity): Pairwise comparison of each dose group with the control
group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urine pH, volume, specific gravity, color and turbidity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. Urine color and turbidity are not evaluated statistically.
Statistics of pathology:
- Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05,
a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Statistics of the clinical examinations:
- Food consumption, body weight and body weight change (parental animals); Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means
Reproductive indices:
For each group, the following calculations were performed:
- Male mating index (%) = (number of males with confirmed mating / number of males placed with females) x 100
- Male fertility index (%) = (number of males proving their fertility / number of males placed with females) x 100
- Female mating index (%) = (number of females mated / number of females placed with males) x 100
- Female fertility index (%) = (number of pregnant females / number of females mated) x 100
- Gestation index (%) = (number of females with live pups on the day of birth / number of pregnant females) x 100
Offspring viability indices:
For each group, the following calculations were performed:
- Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100
- Postimplantation loss (%) = (number of implantations – number of pups delivered / number of implantations) x 100
- Viability index = (Number of live pups on Day 4 post partum / Number of pups born alive) x 100
- Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY:
There were no test substance-related or spontaneous mortalities in any of the groups. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals during the whole study including gestation andlactation periods. Several male and female animals of dose group 3 (1000 mg/kg bw/d) showed salivation after treatment during premating, mating, gestation and lactation. This transient salivation for a few minutes immediately after treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. One sperm positive high-dose female (1000 mg/kg bw/d) and four sperm positive mid-dose females (300 mg/kg bw/d) did not become pregnant.

DETAILED CLINICAL OBSERVATIONS:
Male and female animals of all dose groups (1000, 300 and 100 mg/kg bw/d) did not show any abnormalities.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights and mean body weight change of the male and female F0 generation parental animals in all test substance-treated groups (test groups 1 - 3) were comparable to the concurrent control group during the entire study period. Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (test groups 1 - 3; 100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period.

REPRODUCTIVE PERFORMANCE:
- Males; For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One high-dose male (1000 mg/kg bw/d - No. 38) and four mid-dose males (300 mg/kg bw/d ) did not generate F1 pups. Thus, the male fertility index ranged between 60% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rat of test groups 3 did not show relevant gross lesions.
- Female; The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) varied between 2.4 and 3.7 days without any relation to dosing. All sperm positive rats delivered pups or had implants in utero with the following exceptions: one female in high dose and 4 females in the middle dose. The fertility index varied between 60% in test group 2, 90% in test group 3 and 100% in test group 1 and control. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. None of the non-pregnant females had any relevant gross lesions. The mean duration of gestation was similar in all test groups (i.e. between 22.1 and 22.3 days). The gestation index was 100% in all test groups. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (12.4 / 13.4 / 11.0 and 12.9 implants/dam in test groups 0-3 (0, 100, 300 and 1000 mg/kg body weight/day)). There were no statistically significant differences in post-implantation loss between the groups (5.7% / 5.7% / 7.8% / 5.5%), and the mean number of F1 pups delivered per dam remained unaffected (11.7 / 12.6 / 10.2 and 12.2 pups/dam at 0, 100, 300 and 1000 mg/kg bw/d). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% (test group 3, 2 and control) and 99.2% (test group 2). Moreover, the number of stillborn pups was comparable between the groups.

ORGAN WEIGHTS:
- Absolute weights: When compared to control group 0 (set to 100%), the mean absolute weight of the brain was significantly changed in test group 1 and 2. The significant changes observed in the brain of males showed no dose-dependency and were therefore regarded as non-treatment related. All other mean absolute weight parameters of treated males and females showed no relevant differences when compared to the control group 0 and are therefore considered to be within the normal range.
- Relative weights: All mean relative organ weights of treated males and females showed no statistically significant differences when compared to the control group 0 and are therefore considered to be within the normal range.

GROSS PATHOLOGY:
All gross findings noted at necropsy are regarded as incidental and spontaneous in nature and are not related to treatment.

HISTOPATHOLOGY:
No treatment-related findings were observed in males and females of test group 3 (1000mg/kg bw/d). All findings noted were either single observations, or were biologically equally distributed between control and treated rats. All of them are considered to be incidental and/or spontaneous in origin.

HAEMATOLOGY:
No treatment-related changes among hematological parameters were observed. In males of test group 3 (1000 mg/kg bw/d) absolute and relative basophil counts were increased. However, mean values were within historical control ranges (absolute basophil counts 0.00-0.07 Giga/L; relative basophil counts 0.0-1.1 %). This, were the only changed clinical pathology parameters in these individuals. Therefore, these alterations were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY:
No treatment-related changes among clinical chemistry parameters were observed. In females of test groups 1 and 2 (100 and 300 mg/kg bw/d), urea levels were higher compared to controls. However, the parameter was not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.

URINALYSIS:
No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR:
- Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
- Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
- Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental.
- Quantitative Parameters: No test substance-related impaired parameters were observed in male and female animals of all test groups. The statistically significantly increased distance in the landing foot splay test in high-dosed males (test group 3 - 1000 mg/kg bw/d) was considered to be spontaneous in nature and not treatment related.
- Motor activity measurement (MA): No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all dose groups in comparison to the concurrent control group.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at the highest dose tested.
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at the highest dose tested.

Results: F1 generation

Details on results (F1)

VIABILITY/MORTALITY:
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 99.1% (test group 3), 98.6% (test group 2), 100% (test group 1) and 98.6% (control) without showing any association to the treatment.

CLINICAL SIGNS:
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

BODY WEIGHT:
Mean body weights and mean body weight change of the male and female pups in all test substance-treated groups (test groups 1 - 3) were comparable to the concurrent control group during the entire study period. Two female runts were seen in test group 3 (1000 mg/kg bw/d) and two female runts were seen in test group 1 (100 mg/kg bw/d).

SEX RATIO:
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

GROSS PATHOLOGY:
A few pups showed spontaneous findings at gross necropsy, such as post mortem autolysis and testis discolored (red). These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.



Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at the highest dose tested.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion